EP0077109A2 - DNS Moleküle enthaltend die Gene für Preprochymosin und dessen Reifungsformen und dadurch transformierte Mikroorganismen - Google Patents

DNS Moleküle enthaltend die Gene für Preprochymosin und dessen Reifungsformen und dadurch transformierte Mikroorganismen Download PDF

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EP0077109A2
EP0077109A2 EP82201272A EP82201272A EP0077109A2 EP 0077109 A2 EP0077109 A2 EP 0077109A2 EP 82201272 A EP82201272 A EP 82201272A EP 82201272 A EP82201272 A EP 82201272A EP 0077109 A2 EP0077109 A2 EP 0077109A2
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dna
pur
preprochymosin
regulon
recombinant plasmid
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EP0077109B1 (de
EP0077109A3 (en
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Jan Maat
Cornelis Theodorus Verrips
Adrianus Marinus Ledeboer
Luppo Edens
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Unilever PLC
Unilever NV
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast

Definitions

  • the present invention relates to recombinant DNA and plasmids comprising specific structural genes of mammalian origin coding for the various allelic and maturation forms of preprochymosin, particularly those of bovine origin, and the use of said recombinant plasmids to transform microorganisms in which said genes are expressed.
  • Chymosin is a protein originating from the stomach of newborn mammals.
  • the bovine type (EC 3.4.23.4) is secreted as an inactive precursor, prochymosin, which consists of a single polypeptide chain of 365 amino acid residues (B. Foltmann et al.; Proc. Natl. Acad. Sci. USA; 74, 2321-2324, 1977; B. Foltmann et al.; J. Biol. Chemistry; 254, 8447-8456, 1979).
  • the precursor of bovine prochymosin, preprochymosin consists of a single peptide chain of 381 amino acid residues and differs from prochymosin in an amino-terminal of 16 amino acids. This extension very much resembles a signal sequence which is involved in the process of cotranslational excretion (G.Blobel & D.Dobberstein, J. Cell. Biol. 67, 835-851, 1975).
  • Prochymosin is irreversibly converted into active enzyme (chymosin) by limited proteolysis, during which a total of 42 amino acid residues are released from the amino-terminal part of the peptide chain.
  • the activation is effected through a pH-dependent two-step autocatalytic conversion.
  • the intermediate, pseudochymosin is formed by proteolytic cleavage of bond 27-28 at pH 2-3.
  • the final product, chymosin is formed by activation of pseudochymosin at pH 4-5.
  • the enzymatic activity of chymosin consists of the specific proteolysis of ⁇ -casein. This property makes chymosin widely used as milk-clotting enzyme in cheese manufacture.
  • Chymosin is the essential milk-clotting component of rennet, the crude extract of the abomasum of bovine calves. Rennet is used in the production of several types of cheeses in almost all parts of the world. Because of the ever increasing shortage of calf rennet created by the increasing demand for cheese, many laboratories have been searching for substistutes of microbial origin. Many microbial proteases have been screened, only a few could be used in cheese making, and even these substitutes exhibit a different specificity and therefore may cause an unacceptable texture and/or bitter taste of the cheese. So the production of chymosin by recombinant DNA-containing micro- organisms is expected to become of great economical importance.
  • nucleotide sequences are mainly used to specify the amino acid sequences of proteins that comprise the organism. Although most of the proteins of different organisms differ from each other, the coding relationship between nucleotide sequence and amino acid sequence is fundamentally the same for all organisms.
  • the same nucleotide sequence with cedes for the amino acid sequence of HGH in human pituitary cells will, when transferred to a microorganism, be recognized as coding for the same amino acid sequence.
  • the present invention use is made of recombinant DNA and other molecular biological techniques to construct recombinant DNA molecules that fulfil the above-described requirements.
  • the present invention is also related to the change of the genetic information of structural genes using site-directed mutagenesis.
  • Fig.9 Another preferred inducible regulon is a constituent of the tryptophan system described by F. Lee et al., J. Mol. Biol. 121, 193-217 (1978) and K. Bertrand et al., Science 189, 22-26 (1975).
  • Applicants have modified this tryptophan system to obtain a more adequate system according to Fig.9 .
  • the information coding for the trp attenuator protein is eliminated while maintaining its ribosome-binding site. Expression is highly increased when two trp regulons in a head to tail fashion are being used. Synthesis of this system is illustrated in Fig. 11.
  • the recombinant plasmid according to the invention may comprise DNA sequences which regulate the expression of the structural genes, preferably consisting of a modified promoter/ribosome-binding site of gene VIII of bacteriophage M13, fd or fl (P.M.G.F. van Wezenbeek et al., Gene 11, 129-148 (1980)).
  • N is e.g. represented by A
  • N' should be represented by the complementary base T.
  • the microbial cloning vehicles containing the structural genes encoding the various allelic and maturation forms of preprochymosin according to the invention are produced by a number of steps, the most essential of which are:
  • the fourth stomach of a preruminant calf was ground under liquid nitrogen, extracted with phenol and a selective precipitation of the RNAs with LiCl was performed following the procedure described by K.S. Kirby, Biochem. J. 96, 266-269 (1965), U. Wiegers & H. Hilz (FEBS. Letters, 23, 77-82 (1972). PolyA-containing mRNA was recovered by several passages over oligo-dT-cellulose columns as described by H. Aviv & P. Leder (Proc. Natl. Acad. Sci. USA, 69, 1408-1412 (1972)).
  • Purified (prepro)chymosin mRNA was copied with AMV reverse transcriptase to yield a single-stranded DNA molecule, according to the procedure described by G.N. Buell et al., J. Biol. Chem. 253, 2471-2482 (1978).
  • This cDNA was subsequently converted into a double-stranded molecule using DNA-polymerase, according to the procedure described by A.R. Davis et al., Gene 10, 205-218 (1980). Thereafter the loop structure of the double-stranded DNA copy was removed by SI-nuclease digestion.
  • DNA molecules of the desired length were obtained by polyacrylamide gel-electrophoresis, extracted from the gel and tailed with poly-dC by terminal transferase according to the procedure described by R. Roychoudhury et al., Nucleic Acids Research 3, 863-877 (1976).
  • Plasmid pBR 322 was treated with restriction endonuclease PstI, that cleaves the plasmid at a recognition site that lies in the gene encoding the ampicillin resistance, whereafter the linearized DNA of pBR 322 was supplied at the PstI-site with poly-dG-tails by terminal transferase.
  • the poly-dC-tailed DNA molecules were annealed to the poly-dG-tailed plasmid pBR 322.
  • the plasmids thus obtained were transferred into CaC1 2 -treated E. coli cells. After transformation, cells containing hybrid plasmid DNA molecules were selected on their resistance to tetracycline. (M. Mandel & A. Higa, J. Mol. Biol., 53, 159-162 (1970).
  • the nucleotide sequence analysis of the (prepro)chymosin inserts was performed by the chemical degradation procedure as outlined by A.M. Maxam & W. Gilbert in Methods in Enzymology, L. Grossman & K. Moldave editors, New York, Acad. Press, 1980, Vol. 65 (1), pages 499-560, and the dideoxy/nick translation procedure as outlined by J. Maat & A.J.H. Smith, Nucleic Acid Research, 5, 4537-4545 (1978). Further information on the nucleotide sequence of the (prepro)chymosin .
  • mRNA was derived indirectly by primed synthesis by AMV-reverse transcriptase on the (prepro)chymosin mRNA template in the presence of chain-terminating inhibitors, as outlined by D. Zimmern & P. Kaesberg, Proc. Natl. Acad. Sci. U.S.A. 75, 4257-4261 (1978). This screening yielded inter alia plasmid pUR 1001 containing an almost complete copy of preprochymosin mRNA.
  • Plasmid pBR 322 was cleaved with the restriction enzyme HaeIII and the resulting fragments were subsequently blunt-end ligated with synthetic HindIII-linkers (5') dCCAAGCTTGG (3'). The mixture was subsequently incubated with HindIII and phosphatase. The reactions were terminated by protein extraction with phenol/chloroform (50/50 v/v) and the DNA was then cleaved with PstI. The resulting mixture was subjected to polyacrylamide gel electrophoresis and a 148 bp fragment (fragment A, Fig.
  • Plasmid pUR 1001 was cleaved with EcoRI and treated with calf phosphatase. The mixture was extracted with phenol/chloroform and subsequently PstI was added. Resulting fragments were separated by agarose electrophoresis. Fragment B (see Fig. 3) extending from the EcoRI-site at position 549 to the PstI-site in the noncoding sequence of the preprochymosin gene at the carboxy terminal end of the pUR 1001 clone, was isolated.
  • Fragments A and B were ligated with the large EcoRI-HindIII fragment of pUR 201, 301, 401, 303, 210, 310, 410, 311 (combined called fragment C) yielding pUR 1520, 1530, 1540, 1730, 1820, 1830, 1840, 1930, respectively.
  • plasmid pUR 1001 DNA was cleaved with PstI and resulting DNA fragments were ligated to the synthetic pentanucleotide (5') d HO CTGCA (3'). Following ligation, the mixture was incubated with L. coli DNA polymerase, large fragment, in the presence of dGTP in order to make blunt ends.
  • the DNA was then treated with EcoRI and resulted in fragment (I) of circa 400 bp in length, which was isolated (Fig. 4).
  • Plasmid pUR 1001 was cleaved with PstI and the 1300 bp PstI insert was isolated. This DNA fragment was heat-denaturated to produce single strands.
  • the synthetic primer (5') dGGGGAGGTGG (3') was used to produce complementary DNA synthesis starting from position 198 in the direction of the carboxy terminus by the action of EcoRI DNA polymerase, large fragment. Subsequently the DNA was treated with nuclease Sl to procure blunt-ended dsDNA. To this dsDNA the synthetic EcoRI-linker (5') dCAT(N) n GAATTC(N') n ATG (3') was ligated. After digestion with EcoRI and treatment with phosphatase, the DNA was cleaved once more by BgIII. The resulting fragment II (Fig. 5) was isolated.
  • Plasmid pUR 1001 was treated with HphI, followed by nuclease S1. A 202 base pair long fragment III was isolated (Fig. 6). Fragment III was then ligated to the synthetic EcoRI-linker (5') dCAT(N) n GAATTC(N') n ATG, cleaved with EcoRI and dephosphorylated with phosphatase. The resulting DNA was digested once more by BglII and the resulting fragment IV was isolated.
  • Plasmid pUR 1001 was cleaved with PstI and EcoRI. A fragment of 396 bp was isolated. This fragment was then treated with exonuclease III to procure single-stranded non-complementary DNA (A.J.H. Smith (1979), Nucleic Acids Res. 5, 831-841).
  • This DNA was hybridized to preprochymosin mRNA under conditions described by G. Akusjarvi and U. Petterson (1978), Proc. Natl. Acad. Sci. U.S.A., 75, 5822-5826.
  • the cDNA synthesis was performed as described under 2. Following heat denaturation, dsDNA was made using DNA polymerase, large fragment, with (5') dAGGTGTCTCG OH (3') acting as a primer.
  • the dsDNA was treated with nuclease S1 and ligated to the synthetic EcoRI-linker dCAT(N) n GAATTC(N') n ATG. After EcoRI cleavage and dephosphorylation with (calf intestinal) phosphatase, the DNA was split once more with BgIII. The resulting circa 230 bp long fragment (V) was isolated.
  • a fragment containing 285 base pairs comprising double lac regulon was obtained by restriction endonuclease EcoRI cleavage of pKB 268, described by K. Backman & M. Ptashne, Cell 13, 65-71 (1978). This fragment was ligated in the EcoRI-site of pBR 322 DNA. Plasmid DNA with the lac regulon in the right orientation, pUR 200, (Fig. 8) was partly cleaved by EcoRI in the presence of E. coli RNA polymerase. The EcoRI cleavage site most distant from the restriction endonuclease HindIII-cleavage site was preferentially attacked.
  • the linearized DNA was treated with Sl nuclease, purified by agarose gel electrophoresis, circularized by ligation with T4 DNA-ligase and subsequently transformed into E. coli. From the tetracycline-resistant transformants pUR 201 with the correct structure (Fig. 8) was obtained.
  • a DNA fragment of about 510 base pairs containing the tr p regulon was obtained by restriction endonuclease HinfI cleavage of ptrp ED5, as described by R.A. Hallewell & S. Emtage, Gene 9, 27-47 (1980). This fragment was cleaved with restriction endonuclease TaqI in the presence of E. coLi RNA polymerase.
  • the TaqI-site in the trp regulon (described by K. Bertrand et al., Science 189, 22-26 (1975) and F. Lee et aZ., J. Mol. Biol.
  • Plasmid pUR 300 with the trp regulon in the correct orientation (Fig. 9) was isolated.
  • the EcoRI-cleavage site most distant from the HindIII-site was removed by partial cleavage of pUR 300 DNA by EcoRI in the presence of ethidium bromide and Sl nuclease treatment.
  • Linear DNA molecules were recircularized by T4 DNA ligase. From the tetracycline-resistant transformants pUR 301 with the structure as outlined in Fig. 9 was obtained.
  • a 269 base pairs fragment comprising the gene VIII-promotor was obtained by digestion of RF M13 DNA (DNA sequence 1128-1379 see P.M.G.F. van Wezenbeek et aZ., Gene 11, 129-148 (1980), with the restriction nucleases TaqI and HaeIII; the TaqI-site was made blunt-ended by a repair reaction with E. coZi DNA polymerase; the fragment was subsequently partly digested wi'th restriction enzyme MnII.
  • Plasmid pUR 300 (9b, Fig. 9) was digested with EcoRI and the 234 bp fragment comprising the trp regulon was isolated. This fragment was ligated to pUR 301 DNA, which previously had been cleaved with EcoRI and dephosphorylated with phosphatase. The ligation mixture was used to transform competent E. coli cells and from the ampicillin-resistant transformants pUR 302 was obtained. This plasmid comprises two trp regulons with identical transcription polarity (Fig. 11).
  • pUR 302 was partially cleaved with EcoRI, in the presence of ethidium bromide, treated with nuclease Sl to generate blunt-ends and the cleaved plasmid DNA's were religated.
  • the ligation mix was used to transform competent E . coZ: cells and from the ampicillin-resistant transformants pUR 303 was isolated, wherein the EcoRI-site in between the two trp regulons in pUR 302 had been removed.
  • Plasmid pBR 322 was cleaved with PstI and PvuII, and subsequently dephosphorylated with phosphatase.
  • the 2817 bp long fragment (D, Fig. 12) was isolated.
  • Plasmid pBR 322 was also cleaved with MboII, treated with nuclease S1 and phosphatase and then cleaved once more with PstI.
  • the 400 bp long fragment (E, Fig. 12) extending from position 3201-3608 (J.G. Sutcliffe, Cold Spring Harbor Symposia on Quantitative Biology, 43, 77-90 (1978) was isolated.
  • Plasmid pVU 208 (A.R. Stuitje, thesis, V.U. Amsterdam (1981)) was cleaved with BamHI and treated with nuclease S1.
  • fragments D and E were ligated first, followed by ligation with fragment F.
  • the ligation-mix was used to transform competent E. coli cells. From ampicillin- and tetracycline-resistant transformants pUR 10-containing cells were isolated.
  • the replication- origin-containing fragment is oriented such that the unidirectional replication is in a counter-clockwise direction.
  • plasmids are derived from pUR 201, pUR 301, pUR 303 and pUR 401, respectively, and contain the cop ts replication origin of pUR 10.
  • pUR 10 was digested with PstI and BamHI and the 2841 bp long fragment (G, Fig. 13) was isolated by agarose get electrophoresis and electroelution.
  • Each of the plasmids pUR 201, pUR 301, pUR 303 and pUR 401 was digested with PstI and BamHI and the "regulon"-containing fragments (collectively called H, Fig. 13) were isolated. Fragment G and each of the fragments H in turn were ligated using T4 DNA ligase and the ligation mixes were used to transform competent E. coli cells. From ampicillin- and tetracycline-resistant colonies pU R 210-, pU R 310-, pU R 311- and pU R 410-containing cells were isolated.
  • Plasmids comprising a constitutive or inducible regulon and the ligated preprochymosin gene or its various allelic and maturation forms, the latter being under transcriptional control of said regulons, and transformation of said plasmids into E. coli
  • Plasmids pUR 1520, 1530, 1540, 1730, 1820, 1830, 1840 and 1930 were cleaved with EcoRI and dephosphorylated. Each preparation in turn was ligated with fragment I (8a, Fig. 4). This ligation mix was used to transform competent E. coli RRI and from the ampicillin resistant transformants, cells containing pUR 1521, 1531, 1541, 1731, 1821, 1831, 1841 and 1931 were selected which contained fragment I inserted such that the genetic information coding for pseudochymosin was present as a continuous uninterrupted entity.
  • Plasmid pUR 1521 was cleaved with HindIII, dephosphorylated and then cleaved once more with BglII. The resulting ⁇ 1300 bp long fragment VI (Fig. 15) was purified.
  • the vector fragments C (8a, Fig. 3), in turn, were ligated with fragments II (8b, Fig. 5) and fragment VI.
  • the ligation mix was used to transform competent E. coli cells and from ampicillin-resistant transformants cells containing pUR 1522, 1532, 1542, 1732, 1822, 1832, 1842 and 1932 were selected.
  • Vector fragments C (8a, Fig. 3), fragment IV (8c, Fig. 6) and fragment VI (9b, Fig. 15) were lighted and the resulting ligation mix was used to transform competent E. coli cells. From the amplicillin-resistant transformant cells containing pUR 1523, 1533, 1543, 1733, 1823, 1833, 1843 and 1933 were selected.
  • Vector fragments C (8a, Fig. 3), fragment V (8d, Fig. 7) and fragment VI (9b, Fig. 15) were ligated and the resulting ligation mix was used to transform competent E. coli cells. From the ampicillin-resistant transformants cells containing pUR 1524, 1534, 1544, 1734, 1824, 1834, 1844 and 1934 were selected (Fig. 17).
  • Plasmid pUR 1001 was cleaved with PstI and the resulting DNA fragments were ligated to the synthetic pentanucleotide (5') HO dCTGCA OH (3'). Following ligation, the mixture was incubated with E.
  • M13 1020 contained the coding strand (plus strand); M12 1021 contained the non-coding strand (minus strand).
  • ss.Phage DNA of M13 1020 was converted into double-stranded DNA using E. coli DNA polymerase large fragment, dNTP's and (5') dTGGCCATCCCTGTCC (3') i (675) or (5') dAAACTCATCGTACTG (3') ii (928) in turn as primers, using procedures described by S. Gillam et al. (1979); Nucleic Acids Res., 6, 2973-2985.
  • underlined bases represent mismatches in the primer/template hybrid.
  • phages were screened for the required conversion into the chymosin encoding sequence by plaque hybridization with the 32 p labelled pentadecanucleotides i, ii, as probes and DNA-sequerice analysis.
  • phage isolates M13.1022 and 1023 contained the DNA-sequences: RF M13.1022 and RF M13.1023 were cleaved with EcoRI, dephosphorylated and then cleaved with PstI. From each preparation an 888 bp long fragment was isolated by agarose gel electrophoresis and electroelution. Apart from the required mutations, this fragment corresponds with fragment B (ba, Fig. 3).
  • pUR 1521 (ATCC 39120), 1531, 1541, 1731, 1821, 1831, 1841, 1932 pUR 1522, 1532, 1542, 1732, 1822, 1832 (ATCC 39197), 1842, 1932 pUR 1523,1533 (ATCC 39121), 1543, 1733, 1823, 1833, 1843, 1933 pUR 1524, 1534, 1544, 1734 (ATCC 39198), 1824, 1834, 1844, 1944 with or without the AATT-sequence in the linker netween the regulon and the preprochymosin genes or its maturation forms were cultured under optimal conditions for their growth. These culturing conditions vary with the type of plasmid present in the cells, bui a suitable antibiotic (ampicillin) was always present to maintain selection pressure.
  • the cells containing either plasmids pJR 1521, 1531, 1541, 1731, 1821, 1831, 1841, 1931 or pJR 1522, 1532, 1542, 1732, 1822, 1832, 1842, 1932 or pJR 1523, 1533, 1543, 1733, 1823, 1833, 1843, 1933 or pUR 1524, 1534, 1544, 1734, 1824, 1834, 1844, 1944 produced considerable amounts of pseudochymosin, chymosin, prochymosin or preprochymosin, respectively. These amounts varied from 10 3 -10 7 molecules/cell.
  • E. coli cells which contained preprochymosin or modified preprochymosin encoding plasmids contained (modified) preprochymosin in the cytoplasm and prochymosin in their periplasmic space.
  • the bacterially produced preprochymosin, prochymosin and pseudochymosin could be converted into chymosin using the procedures described by V. Barkholt Pedersen et al. (Eur. J. Biochem., 94, 573-580 (1979)).
  • the chymosins which were thus obtained and bacterially-produced chymosin were shown to be fully biologically active in proteolysis.
  • the presence of the proteins was further demonstrated by SDS- polyacrylamide gel electrophoresis with or without immuno- precipitation, and by immunological ELISA and RIA tests.
  • the antisera for this test were generated by injecting bovine calf chymosin supplemented with Freund adjuvant into sheep as well as rabbits.
  • DNA is drawn as a single-lined circle, still this represents double-stranded DNA (bacteriophage M13.
  • DNA is single-stranded; the replicative form RF, however, is double-stranded).
  • 5'-ends of cleaved DNA at restriction enzyme cleavage site are phosphorylated unless indicated otherwise; 3'-ends are always dephosphorylated.
EP19820201272 1981-10-14 1982-10-13 DNS Moleküle enthaltend die Gene für Preprochymosin und dessen Reifungsformen und dadurch transformierte Mikroorganismen Expired EP0077109B1 (de)

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AT82201272T ATE45767T1 (de) 1981-10-14 1982-10-13 Dns molekuele enthaltend die gene fuer preprochymosin und dessen reifungsformen und dadurch transformierte mikroorganismen.

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GB8131004 1981-10-14
GB8131004 1981-10-14

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EP0077109A2 true EP0077109A2 (de) 1983-04-20
EP0077109A3 EP0077109A3 (en) 1983-06-29
EP0077109B1 EP0077109B1 (de) 1989-08-23

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AU (1) AU551251B2 (de)
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0096430A1 (de) 1982-05-19 1983-12-21 Gist-Brocades N.V. Klonierungssystem für kluyveromyce Spezies
FR2529570A1 (fr) * 1982-07-01 1984-01-06 Genex Corp Gene de chymosine ou de prochymosine de veau isole, et son utilisation dans un procede de production d'une chymosine ou prochymosine de veau pratiquement pure
WO1985000382A1 (en) * 1983-07-06 1985-01-31 Gist-Brocades N.V. Molecular cloning and expression in industrial microorganism species
EP0133756A2 (de) * 1983-07-06 1985-03-06 Genex Corporation Vektor für die Expression von Polypeptiden
EP0134662A1 (de) * 1983-07-07 1985-03-20 Genex Corporation Herstellung von Rinderkalbschymosin
EP0147178A2 (de) * 1983-12-23 1985-07-03 Pfizer Inc. Expressionsplasmide für die Herstellung eines heterologen Proteins in Bakterien
WO1985005377A1 (en) * 1984-05-11 1985-12-05 Codon Genetic Engineering Laboratories An efficient process for producing active chymosin from a precursor protein synthesized in bacteria
US4721673A (en) * 1983-09-01 1988-01-26 Genex Corporation Recovery and activation process for microbially produced calf prochymosin
GB2200118A (en) * 1987-01-23 1988-07-27 Allelix Inc Synthetic chymosin-and prochymosin-encoding DNA segments
US4801537A (en) * 1984-06-08 1989-01-31 Genex Corporation Vector for expression of polypeptides in bacilli
US4935370A (en) * 1983-12-23 1990-06-19 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
WO1990015860A1 (en) * 1989-06-16 1990-12-27 Genencor, Inc. Dna sequences, vectors, and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
US5082775A (en) * 1984-05-11 1992-01-21 Berlex Laboratories, Inc. Efficient process for isolating insoluble heterologous protein using non-ionic detergents
US5364770A (en) * 1985-08-29 1994-11-15 Genencor International Inc. Heterologous polypeptides expressed in aspergillus
US5496711A (en) * 1981-01-16 1996-03-05 Genome Therapeutics Corp. Processes for producing pre-prorennin, prorennin and rennin
US5679543A (en) * 1985-08-29 1997-10-21 Genencor International, Inc. DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
US5766912A (en) * 1986-03-17 1998-06-16 Novo Nordisk A/S Humicola lipase produced in aspergillus
US5863759A (en) * 1986-03-17 1999-01-26 Novo Nordisk A/S Process for the production of protein products in aspergillus
US6004785A (en) * 1985-08-29 1999-12-21 Genencor International Inc. Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same
US6083718A (en) * 1983-07-06 2000-07-04 Gist-Brocades, N.V. Transformed industrial bacillus strains and methods for making and using them
KR100762768B1 (ko) * 2000-04-14 2007-10-02 퀄컴 인코포레이티드 통신 시스템에서 신호를 고속 재송신하는 방법 및 장치
US7482148B2 (en) * 2004-03-30 2009-01-27 Sudershan Biotech Ltd. Recombinant calf-chymosin and a process for producing the same

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JPS59166086A (ja) * 1983-03-09 1984-09-19 Teruhiko Beppu 新規な発現型プラスミドとそれらを用いて仔牛プロキモシン遺伝子を大腸菌内で発現させる方法
NL8400687A (nl) * 1984-03-02 1985-10-01 Unilever Nv Recombinant plasmiden; recombinant plasmiden bevattende bacterien; werkwijze voor het bereiden of vervaardigen van een zuivelproduct; werkwijze voor het bereiden van een eiwit.

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Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5525484A (en) * 1981-01-16 1996-06-11 Genome Therapeutics Corp. Recombinant DNA means and method for producing rennin, prorenin and pre-prorennin
US5496711A (en) * 1981-01-16 1996-03-05 Genome Therapeutics Corp. Processes for producing pre-prorennin, prorennin and rennin
EP0096430A1 (de) 1982-05-19 1983-12-21 Gist-Brocades N.V. Klonierungssystem für kluyveromyce Spezies
FR2529570A1 (fr) * 1982-07-01 1984-01-06 Genex Corp Gene de chymosine ou de prochymosine de veau isole, et son utilisation dans un procede de production d'une chymosine ou prochymosine de veau pratiquement pure
EP0133756A3 (de) * 1983-07-06 1986-10-01 Genex Corporation Vektor für die Expression von Polypeptiden
US6083718A (en) * 1983-07-06 2000-07-04 Gist-Brocades, N.V. Transformed industrial bacillus strains and methods for making and using them
WO1985000382A1 (en) * 1983-07-06 1985-01-31 Gist-Brocades N.V. Molecular cloning and expression in industrial microorganism species
EP0133756A2 (de) * 1983-07-06 1985-03-06 Genex Corporation Vektor für die Expression von Polypeptiden
EP0134048A1 (de) * 1983-07-06 1985-03-13 Gist-Brocades N.V. Molekulare Klonierung und Expression in industriellen Mikroorganismen
EP0134662A1 (de) * 1983-07-07 1985-03-20 Genex Corporation Herstellung von Rinderkalbschymosin
US4721673A (en) * 1983-09-01 1988-01-26 Genex Corporation Recovery and activation process for microbially produced calf prochymosin
EP0147178A3 (en) * 1983-12-23 1987-06-03 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
US4935370A (en) * 1983-12-23 1990-06-19 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
US5955297A (en) * 1983-12-23 1999-09-21 Pfizer Inc. Expression plasmids for improved production of heterologous protein in bacteria
EP0147178A2 (de) * 1983-12-23 1985-07-03 Pfizer Inc. Expressionsplasmide für die Herstellung eines heterologen Proteins in Bakterien
US5082775A (en) * 1984-05-11 1992-01-21 Berlex Laboratories, Inc. Efficient process for isolating insoluble heterologous protein using non-ionic detergents
WO1985005377A1 (en) * 1984-05-11 1985-12-05 Codon Genetic Engineering Laboratories An efficient process for producing active chymosin from a precursor protein synthesized in bacteria
US4801537A (en) * 1984-06-08 1989-01-31 Genex Corporation Vector for expression of polypeptides in bacilli
US5364770A (en) * 1985-08-29 1994-11-15 Genencor International Inc. Heterologous polypeptides expressed in aspergillus
US6171817B1 (en) 1985-08-29 2001-01-09 Genencor International, Inc. Heterologous polypeptides expressed in filamentous fungi, process for making same, and vectors for making same
US5679543A (en) * 1985-08-29 1997-10-21 Genencor International, Inc. DNA sequences, vectors and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
US6379928B1 (en) 1985-08-29 2002-04-30 Genencor International, Inc. Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same
US5578463A (en) * 1985-08-29 1996-11-26 Genencor International, Inc. Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same
US6103490A (en) * 1985-08-29 2000-08-15 Genencor International, Inc. Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same
US6004785A (en) * 1985-08-29 1999-12-21 Genencor International Inc. Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same
US5965384A (en) * 1986-03-17 1999-10-12 Novo Nordisk A/S Methods for producing Humicola lipases in aspergillus
US5863759A (en) * 1986-03-17 1999-01-26 Novo Nordisk A/S Process for the production of protein products in aspergillus
US5766912A (en) * 1986-03-17 1998-06-16 Novo Nordisk A/S Humicola lipase produced in aspergillus
US7517668B1 (en) 1986-03-17 2009-04-14 Novozymes A/S Process for the production of protein products in aspergillus
GB2200118A (en) * 1987-01-23 1988-07-27 Allelix Inc Synthetic chymosin-and prochymosin-encoding DNA segments
WO1990015860A1 (en) * 1989-06-16 1990-12-27 Genencor, Inc. Dna sequences, vectors, and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi
KR100762768B1 (ko) * 2000-04-14 2007-10-02 퀄컴 인코포레이티드 통신 시스템에서 신호를 고속 재송신하는 방법 및 장치
US7482148B2 (en) * 2004-03-30 2009-01-27 Sudershan Biotech Ltd. Recombinant calf-chymosin and a process for producing the same

Also Published As

Publication number Publication date
JPS58109499A (ja) 1983-06-29
JPH0728740B2 (ja) 1995-04-05
EP0077109B1 (de) 1989-08-23
EP0077109A3 (en) 1983-06-29
AU551251B2 (en) 1986-04-24
DK453782A (da) 1983-04-15
DE3279899D1 (en) 1989-09-28
IE55006B1 (en) 1990-04-25
AU8933182A (en) 1984-04-19
ATE45767T1 (de) 1989-09-15
JPH05192168A (ja) 1993-08-03
DK139493D0 (da) 1993-12-16
BR8205954A (pt) 1983-09-13
IE822479L (en) 1983-04-14
DK139493A (da) 1993-12-16
JPH05219951A (ja) 1993-08-31
JP2645681B2 (ja) 1997-08-25
JPH0471517B2 (de) 1992-11-13

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